Self-assembled, aptamer-tethered DNA nanotrains for targeted transport of molecular drugs in cancer theranostics

Abstract

Nanotechnology has allowed the construction of various nanostructures for applications, including biomedicine. However, a simple target-specific, economical, and biocompatible drug delivery platform with high maximum tolerated doses is still in demand. Here, we report aptamer-tethered DNA nanotrains (aptNTrs) as carriers for targeted drug transport in cancer therapy. Long aptNTrs were self-assembled from only two short DNA upon initiation by modified aptamers, which worked like locomotives guiding nanotrains toward target cancer cells. Meanwhile, tandem "boxcars" served as carriers with high payload capacity of drugs that were transported to target cells and induced selective cytotoxicity. aptNTrs enhanced maximum tolerated dose in nontarget cells. Potent antitumor efficacy and reduced side effects of drugs delivered by biocompatible aptNTrs were demonstrated in a mouse xenograft tumor model. Moreover, fluorophores on nanotrains and drug fluorescence dequenching upon release allowed intracellular signaling of nanotrains and drugs. These results make aptNTrs a promising targeted drug transport platform for cancer theranostics.

Keywords: DNA nanomedicines; in vivo; self-assembly; targeted anticancer drug delivery.

Fig. 1.

Schematics of the self-assembly of aptamer-tethered DNA nanotrains (aptNTrs) for transport of molecular drugs in theranostic applications. (A) Self-assembly of aptNTrs from short DNA building blocks (1) upon initiation from a chimeric aptamer-tethered trigger probe. The resultant long nanotrains (2) were tethered with aptamers working as locomotives on one end, with multiple repetitive “boxcars” to be loaded with molecular drugs (3). AFM images (1–3) showed the morphologies of the corresponding nanostructures [1, M1+M2; 2, sgc8–NTrs; 3, sgc8–NTrs loaded with molecular drugs (Dox)]. (B) The drugs were specifically transported to target cancer cells via aptNTrs, unloaded, and induced cytotoxicity to target cells. The fuorescence of drugs loaded onto nanotrains was quenched (“OFF”), but was recovered upon drug unloading (“ON”), enabling this platform to signal target recognition and drug unloading.

Fig. 2.

Characterization of the formation, selective cancer cell recognition, and internalization of sgc8–NTrs. (A) Agarose gel electrophoresis showing the self-assembly of sgc8–NTrs initiated by sgc8–trigger. (B and C) Flow-cytometric results showing the selective recognition abilities of sgc8–NTrs to CEM cells (B), but not to Ramos cells (C). The presence of multiple monomers on one nanotrain resulted in signal amplification of sgc8–NTr-bound CEM cells [lib, sgc8, M1, M2: labeled with FITC; (M1+M2): unpolymerized M1 and M2]. (D and E) Confocal laser-scanning microscopy images displaying the internalization of sgc8–NTrs into target CEM cells. Cells were incubated with sgc8–NTrs (100 nM sgc8–trigger equivalents) at 37 °C for 2 h, followed by transferrin–Alexa 633 staining. The intracellular TAMRA fluorescence signal (denoted by arrows in D) colocalized with Alexa 633 signal indicates the internalization of sgc8–NTrs through endocytosis (M2: labeled with TAMRA). (Scale bars: D, 100 µm; E, 10 µm.)

Fig. 3.

Targeted drug transport using aptNTrs with high payload capacity and stability. (A) Fluorescence spectra of Dox (2 µM) with increasing equivalents of sgc8–NTrs (shown by values from top to bottom). The fluorescence quenching indicates drug loading into sgc8–NTrs. The apparent Dox fluorescence quenching with as low as 1/50 NTr equivalents reflects high drug payload capacity. (B) Scattered data points showing the fluorescence intensities of Dox diffused from free Dox or sgc8–NTr–Dox (Dox: 30 µM) during dialysis to outside PBS buffer at different time points. Data were fit to a drug release model by nonlinear regression (fit lines shown in solid). The negligible drug diffusion from nanotrains, in contrast to fast diffusion from free Dox, indicates the high stability of sgc8–NTr–Dox complexes. (C–F) Confocal laser-scanning microscopy images displaying the intracellular signaling of drug unloading and selective drug transport to target cells by sgc8–NTrs. Target CEM cells (C and D) and nontarget Ramos cells (E and F) were treated with free Dox (C and E; 2 µM) and sgc8–NTr–Dox (D and F; 2 µM Dox equivalents), followed by transferrin–Alexa 633 staining. The comparable intracellular Dox fluorescence intensities in C and D, but significantly lower intensities in F than E, indicate the selectivity of drug transport via sgc8–NTrs (Insets: enlarged cells). (Scale bar: 100 µm.)

Fig. 4.

Selective cytotoxicity of molecular drugs (Dox) transported by aptNTrs. (A and B) MTS assay results showing that Dox transported by sgc8–NTrs (sgc8–NTr–Dox) selectively induced potent cytotoxicity and inhibited cell proliferation in target CEM cells (A), but not in nontarget Ramos cells (B), in contrast to nonselective cytotoxicity induced by free Dox in both target and nontarget cells.

Fig. 5.

Potent antitumor efficacy and reduced side effects of drugs transported via aptNTrs. CEM xenograft mouse tumor model was developed by s.c. injection of CEM cells in the back of NOD.Cg-Prkdc (scid) IL2 mice. Mice were divided into three groups that are, respectively, treated by i.v. injections of (i) sgc8–NTrs, (ii) free Dox, and (iii) sgc8–NTr–Dox, with 2 mg/kg Dox or Dox equivalent dosages in ii and iii and accordingly 23 mg/kg sgc8–NTrs in i. (A) Tumor volume up to day 10 after treatment initiation (mean ± SD; n = 5). Asterisk on day 10 represents significant differences between tumor volumes of free Dox- and sgc8–NTr–Dox-treated mice (*P < 0.05, n = 5; Student t test). (B) Survival percentage of mice after treatment initiation. (C) Mouse body weight loss at day 10 compared with day 0, after treatment initiation (mean ± SD; n = 5). Asterisk represents significant differences between weight loss of free Dox- and sgc8–NTr–Dox-treated mice (***P < 0.001, n = 5; one-way ANOVA with Newman–Keuls post hoc test).